Basophils are white blood cells that play an important role in the human immune system. These cells physiologically increase in number in immune response to certain allergies, chronic inflammation, and parasitic infections. Basophils are also a significant indicator for the presence of certain malignancies such as chronic myeloproliferative neoplasms and acute myeloid leukemia. In the current manuscript we present a statistically significant correlation between persistent basophilia in primary myelofibrosis (PMF) and the risk for the subsequent development of acute myeloid leukemia. We have retrospectively identified in the files of the Department of Hematology, Ion Chiricuta Clinical Cancer Center in Cluj Napoca, Romania 623 consecutive patients diagnosed with AML over a period spanning from 2008 to 2018. We afterwards identified 32 patients with AML diagnosis following a previous diagnosis of myelofibrosis (either post-PV, post-ET, or post-PMF). All the patients were diagnosed according to the WHO criteria. We subsequently established a control group consisting of 32 patients with underlying BCR-ABL-negative MPN who did not develop AML (AML-negative group). Following this, we assessed whether the AML-negative patients from our control group also had a persistent (>3 months) absolute basophilia. When comparing both groups of patients with myelofibrosis, the group with subsequent AML development and the one without AML, the follow-up did not present statistically significant differences between the two groups. In the univariate analysis, patients who progressed to AML had more frequently basophilia, longer basophilia duration, higher pre-therapy absolute, and relative basophil count and presented more frequently calreticulin (CALR) mutations. In the current study, we emphasize the need for a closer clinical monitoring for chronic MPNs with marked basophilia, with an important potential clinical impact.

Secondary myeloid neoplasia may be a complication of intensive cytostatic therapy. The most common types of secondary neoplasias are acute myeloid leukemia and myelodysplastic syndrome. The development of secondary atypical chronic myeloid leukemia (aCML) is an extremely rare phenomenon. The paper describes transformation of secondary myelodysplastic syndrome to aCML 6 months after its diagnosis. The development of aCML was accompanied by additional chromosomal aberration as monosomy of chromosome 17. No mutations in the JAK2, MPL, and CalR genes were detected. It is concluded that the clinical course of secondary myeloid neoplasias is variable.

Essential thrombocythemia (ET) is a myeloproliferative neoplasm characterized by a clonal expansion of megakaryocytes. ET can result in both arterial and venous thrombosis. Involvement of the coronary arteries has been reported. Patients who harbor a CALR mutation are half as likely to suffer a thrombotic event as compared to patients with a JAK2 mutation. We report a case of CALR-mutated ET whose initial disease manifestation was a non-ST segment elevation myocardial infarction.

BACKGROUND Essential thrombocythemia (ET) is a type of myeloproliferative neoplasm (MPN) characterized by sustained thrombocytosis in peripheral blood. Patients typically have gene mutations like JAK2V617F, CALR, and MPLW515L/K. This report describes a young man with ET without any of the above mutations who had paradoxical bleeding due to acquired Von-Willebrand disease. CASE REPORT A young man with a medical history of thrombocytosis on aspirin presented with acute chest pain and was found to have had a myocardial infarction. Emergency cardiac catheterization revealed a thrombotic occlusion of the left anterior descending (LAD) artery and the right posteriolateral system, with an ejection fraction of 25%. He underwent thrombectomy and balloon angioplasty with LAD stenting, and an Impella 2.5 was inserted due to severe left ventricular dysfunction with akinesia. Aspirin and ticagrelor were administered, but the patient later experienced postoperative bleeding from the site of the Impella device. The bleeding was attributed to acquired Von-Willebrand disease secondary to ET. Emergency plateletpheresis was recommended. Further workup demonstrated that he was triple-negative for JAK2, MPL, and CALR gene mutations. CONCLUSIONS The paradoxical bleeding resulting from acquired Von-Willebrand disease was likely an entirely separate entity from the hyper-thrombotic state expected from ET. Careful assessment of clinical symptoms and laboratory markers, in addition to a high degree of suspicion, are needed to diagnose acquired Von-Willebrand disease as a complication of ET.

Objective: To investigate the gene mutation occurved in AML patients with 29 kinds of fusion genes and 51 kinds of tumor gene.

Methods: Next-generation sequencing (NGS) was used to detected the 49 kinds of targeted gene. FLT3 internal tandem duplication (FLT3-ITD), CALR, NPM1 and CEBPA mutation were detected by DNA-based PCR and Sanger sequencing. Twenty-nine kinds of fusion genes were dected by multiplex nested RT-PCR.

Results: The total gene mutation rate was 91% (109/121) in all the 121 patients. On average, 2.1 mutated genes per patient were identified, among these 121 patients, coexistence of ≥ 3 mutations was frequent (34.7%). The most commonly mutated genes were NRAS (23.96%, n=29), followed by NPM1 (14.04%, n=17), CEBPA double mutations (14.04%, n=17), KRAS (11.57%, n=14)，FLT3-ITD (10.74%, n=13), CSF3R (10.74%, n=13), TET2 (9.92%, n=12) and IDH1 (9.1%, n=11). Overall, fusion genes were detected in 47 (37.3%) patients, including AML/ETO (n=12), CBFβ/MYH11 (n=11), PML/RARa (n=12), MLL rearranagement realated mutation MLL-X (n=10). TLS/ERG (n=1) and DEK/CAN (n=1) in an order of decreasing frequency. Patients with normal karyotype (NK)- AML exhibited more mutations in CEBPA, NPM1, TET2, RUNX1 and IDH1, comparing with abnormal karyotype patients. KRAS mutation in abnormal kayotype patients was significantly higher than that in normal kayotype patients (P=0.014). TP53 mutations were predominantly associated with complex cytogenetics (P=0.199). KRAS mutations were more frequent in core binding factor (CBF) acute myeloid leukemia (AML) and 11q23/MLL rearrangement leukemia, compared with NK-AML (P=0.006 and 0.003, respectively). KIT mutations predominated in CBF-AML (P=0.006). JAK2V617F mutations were detected in two patients and co-occurred with AML-ETO fusions.

Conclusion: At least one mutation is observed in more than 90% patients. On average, more than 2 mutated genes per patient are identified. Some gene mutations are associated with gene rearrangement.

题目: 121例急性髓系白血病患者基因突变谱的分析.

目的: 研究急性髓系白血病（AML）患者29种融合基因及51种肿瘤基因突变的发生情况.

方法: 采用第二代DNA测序技术检测49种靶基因；采用DNA-PCR检测FLT3-ITD、NPM1基因12号外显子、CALR基因9号外显子及CEBPA的TAD、BZIP 2个功能区的突变发生情况；采用多重PCR法检测29种白血病融合基因.

结果: 121例患者中，基因突变总发生率为90.1%（109/121），每例患者平均发生2.1个基因突变，34.7%的患者同时携带≥3个基因突变。突变检出率最高的基因为NRAS（23.96%，n=29），其他基因依次为：NPM1（14.04%，n=17）、CEBPA双突变（14.04%，n=17）、KRAS（11.57%，n=14）、FLT3-ITD（10.74%，n=13）、CSF3R（10.74%，n=13）、TET2（9.92%，n=12）和IDH1（9.1%，n=11）。共47例患者检出融合基因，包括AML/ETO 12例，CBFβ/MYH11 11例，PML/RARa 12例，涉及MLL重排的MLL-X融合基因10例，TLS/ERG 1例及DEK/CAN 1例。 CEBPA、NPM1、TET2、RUNX1及IDH1突变在正常核型中的检出率均高于异常核型，差异有统计学意义；KRAS突变在异常核型中的发生率高于正常核型，差异显著（P=0.014）。复杂核型患者的TP53突变检出率明显高于正常核型及其他异常核型（P=0.199）。KRAS突变在核心结合因子白血病及伴有11q23/MLL重排的M5患者中的阳性率均高于正常核型（P=0.006，P=0.003）。KIT基因突变主要见于核心结合因子白血病，两者有相关性（P=0.006）。共检测到2例JAK2V617F突变，均与t (8; 21)/AML1/ETO伴随出现.

结论: 大于90%的AML患者至少携带1个基因突变，每例患者平均发生2个以上白血病常见致病基因突变，部分基因突变与基因重排有一定相关性.

Myeloproliferative neoplasms (MPNs) are classified as chronic myeloid leukemia (CML) and Philadelphia chromosome-negative MPN. In MPN cases, the presence of a BCR-ABL1 translocation with a coexisting mutation is exceptionally rare. Herein, we report the first documented patient with CML harboring CALR mutation in Korea. A 33-year-old woman was referred to our hospital in February 2015 with splenomegaly, leukocytosis, and thrombocytosis. She was diagnosed with CML and started receiving nilotinib. In October 2015, a major molecular response was observed, but thrombocytosis persisted. A repeat bone marrow (BM) examination revealed no specific findings. However, as thrombocytosis worsened, we changed nilotinib to dasatinib. In May 2019, owing to persistent thrombocytosis, we repeated the BM examination and found CALR mutation (15.97%) on the MPN-next generation sequencing (NGS) test. We then retrospectively performed repeat MPN-NGS testing using the BM aspirate sample obtained in 2015 and found CALR mutation (10.64%).

OBJECTIVE

Calreticulin (CALR) plays important roles in cell proliferation, apoptosis, and immune responses. CALR mutations were described recently in Janus kinase 2 gene (JAK2)-negative or MPL-negative primary myelofibrosis (PMF) and essential thrombocythemia (ET) patients. CALR trails JAK2 as the second most mutated gene in myeloproliferative neoplasms (MPNs). However, little is known about CALR mutation in Chinese patients with leukemia. In the present study, a cohort of 305 Chinese patients with hematopoietic neoplasms was screened for CALR mutations, with the aim of uncovering the frequency of CALR mutations in leukemia and MPNs.

METHODS

Polymerase chain reaction and direct sequencing were performed to analyze mutations of CALR in 305 patients with hematopoietic malignancies, including 135 acute myeloid leukemia patients, 57 acute lymphoblastic leukemia patients, and 113 MPN patients.

RESULTS

CALR mutations were found in 10.6% (12 of 113) of samples from patients with MPNs. CALR mutations were determined in 11.3% (6 of 53), 21.7% (5 of 23), and 9.1% (1/11) of patients with ET, PMF, and unclassifiable MPN, respectively.

CONCLUSIONS

We showed that MPN patients carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels compared to those with mutated JAK2. However, all of the leukemia patients had negative results for CALR mutations.

The development of acute lymphoblastic leukemia in an existing myeloproliferative neoplasm is rare with historical cases unable to differentiate between concomitant malignancies or leukemic transformation. Molecular studies of coexisting JAK2 V617F-positive myeloproliferative neoplasms and mature B cell malignancies indicate distinct disease entities arising in myeloid and lymphoid committed hematopoietic progenitor cells, respectively. Mutations of CALR in essential thrombocythemia appear to be associated with a distinct phenotype and a lower risk of thrombosis yet their impact on disease progression is less well defined. The as yet undescribed scenario of pro-B cell acute lymphoblastic leukemia arising in CALR mutated essential thrombocythemia is presented. Intensive treatment for the leukemia allowed for expansion of the original CALR mutated clone. Whether CALR mutations in myeloproliferative neoplasms predispose to the acquisition of additional malignancies, particularly lymphoproliferative disorders, is not yet known.

No specific translocation is associated with myeloproliferative neoplasms (MPNs). However, an interstitial deletion involving subband 17q11.2 which includes the NF1 gene, although rare, is a recurrent aberration in several myeloid disorders including MPNs. For the first time, we report an acquired novel translocation involving 10p13 and 17q11.2 in a 62-year-old Caucasian female which was referred for investigation of chronic and persistent unexplained thrombocytosis. The patient had no history of hematological sequelae and genomic testing for JAK2, CALR, and MPL mutations were negative. She was subsequently diagnosed with a triple negative essential thrombocythemia. Array-CGH analysis noted that the translocation harbored two cryptic deletions, one of which involved 17q11.2 encompassing the NF1 gene. One of the junction breakpoints involved the SUZ12 gene. Immunohistochemical assessment of the marrow trephine showed increased megakaryocytic expression of the SUZ12 protein, as well as EZH2 and Ki67; biochemical abnormalities suggestive of excess megakaryocytic hyperplasia. This novel translocation may affect the expression of SUZ12 and its downstream targets, and may represent a unique pathogenomic etiology which drives chronic thrombocytosis in essential thrombocythemia.

Keywords: NF1; SUZ12; chronic thrombocytosis; deletion 17q11.2; essential thrombocythemia; t(10;17)(p13;q11.2).

In our previous study, five different secretory proteins, including GSN, ADAMTSL4, CALR, PPIA and TXN, have been identified to be associated with the nasopharyngeal carcinoma (NPC) metastasis.In this work, the 5 proteins were further investigated. Bioinformatics analysis suggested that they might play an important role in the process of NPC development.Western blotting analysis showed that all of these 5 targets could be secreted into extracellular by both high metastatic NPC 5-8F cells and non-metastatic NPC 6-10B cells. Except for GSN, the expressions of ADAMTSL4, CALR, PPIA and TXN proteins in extracts of the 5-8F and 6-10B cells were significantly different (P < 0.05). Thus, the expressions of these 4 differentially expressed proteins were further tested in a cohort of NPC tissue specimens. The results indicated that the expression levels of ADAMTSL4 and TXN were highly correlated with the lymph node and distant metastasis (P<0.05) in NPC patients. Moreover, Enzyme-linked immunosorbent assay (ELISA) was used to investigate the concentrations of the ADAMTSL4 and TXN in serum specimens of NPC patients. The results revealed that serum ADAMTSL4 expression level was closely correlated with lymph node metastasis and clinical stage (P<0.05) in NPC patients, and it was able to discriminate metastasis NPC from non-metastasis NPC with a sensitivity of 75.6% and a specificity of 64.7%. The present data show for the first time that the ADAMTSL4 and TXN may be novel and potential biomarkers for predicting the NPC metastasis.Furthermore, the serum ADAMTSL4 could be a potential serum tumor biomarker for prognosis of NPC.

BACKGROUND

This study aimed to determine GATA1 expression levels to better characterize subgroups in BCR/ABL1-negative myeloproliferative neoplasms (MPNs).

METHODS

This study enrolled 49 patients diagnosed as having BCR/ABL1-negative MPN on the basis of the 2016 World Health Organization classification : nine polycythemia vera (PV), 17 essential thrombocythemia (ET), 12 prefibrotic primary myelofibrosis (prePMF), and 11 overt primary myelofibrosis (PMF). Relevant clinical and laboratory data were retrieved from the medical records. The molecular analysis of CALR and MPL mutations and quantification of JAK2 V617F allele burden were performed. GATA1 expression was assessed by an immunohistochemical assay on bone marrow biopsy. GATA1 expression was analyzed serially in 18 patients.

RESULTS

GATA1 expression decreased significantly in PMF compared with that in other subtypes, while no statistical difference was identified between ET and prePMF. GATA1 expression did not differ according to the mutation profiles or the allele burden of JAK2 V617F, but it decreased significantly in patients with overt fibrosis or leukemic transformation.

CONCLUSIONS

Our results suggest that GATA1 expression is significantly low in PMF and decreases with progressive fibrosis and possibly with leukemic transformation, although our attempt to accurately distinguish between subgroups using GATA1 immunohistochemical approach did not achieve statistical significance. A large patient cohort with long term follow-up is required to evaluate the prognostic value of GATA1 expression.

Primary myelofibrosis (PMF) is a Philadelphia chromosome negative, clonal myeloproliferative neoplasm characterised by a progressive nature. Morphologically, the bone marrow biopsy shows features of abnormal proliferation of terminally differentiated megakaryocytes and subsequent bone marrow fibrosis. The molecular landscape of PMF includes phenotypic driver mutations (JAK2 V617F, CALR and MPL) which represent major diagnostic criteria, and subclonal mutations that also occur in several other myeloid diseases, but have a prognostic value in disease progression of MF. The most important subclonal mutations affect the genes ASXL1, TET2, IDH1/2, EZH2 and TP53. Triple negative genotype and the high molecular risk genotype and CALR-/ASXL1+ are associated with adverse survival with the latest indicating stem cell transplantation independently of the DIPSS-plus score.

A 83-year-old female patient was admitted to our hospital due to hematological manifestation of juvenile granulocytes and macrocytic anemia. Bone marrow (BM) examination revealed erythroid dysplasia and cytoplasmic blasts, and hence the patient was diagnosed with myelodysplastic syndrome with ring sideroblasts and with single lineage dysplasia (MDS-RS-SLD). Erythrocyte transfusion was performed as a supportive therapy, and there was a gradual increase in the number of blood cells. Therefore, BM re-examination was performed and it was confirmed that the number of megakaryocytes increased, so the patient's condition was determined as myelodysplastic/myeloproliferative neoplasms with ring sideroblasts and thrombocytosis (MDS/MPN with RS-T). Incidentally, gene mutation analysis showed CALR gene mutation. Thereafter, administration of hydroxycarbamide and anagrelide did not show adverse events and complications, and a good blood count control was obtained. Furthermore, it was also confirmed that an SF3B1 gene mutation is highly positive in MDS-RS. There was no report on CALR-mutant MDS/MPN in Japan, and it is a rare disease overseas.

The Wilms tumor gene WT1 is a useful marker of clonal hematopoiesis and it has been shown to be a good marker of residual disease and it reflects the response to therapy. Although myelofibrosis is characterized by mutations of JAK2 and calreticulin (CALR), these mutations are not useful to monitor response to therapy. In this study we demonstrated that in patients affected by myelofibrosis WT1 correlates with the International Prognostic Scoring System (IPSS) score at diagnosis. Furthermore WT1 is a good marker of response to JAK2 inhibitors especially for patients without blasts and for patients who develop anemia or thrombocytopenia not for progression but as therapy related toxicity. Finally, WT1 transcript reduction can mirror a benefit of therapy on the disease burden. This study demonstrated that WT1 is a good marker for monitoring the response to therapy in patients affected by myelofibrosis.

Noroviruses are the primary pathogens associated with shellfish-borne gastroenteritis outbreaks. These viruses remain stable in oysters, suggesting an active mechanism for virus concentration. In this study, a deep RNA sequencing technique was used to analyze the transcriptome profiles of Pacific oysters at different time points after inoculation with norovirus (GII.4). We obtained a maximum of 65, 294, 698 clean sample reads. When aligned to the reference genome, the average mapping ratio of clean data was approximately 65%. In the samples harvested at 12, 24, and 48 h after contamination, 2,223, 2,990, and 2020 genes, respectively, were differentially expressed in contaminated and non-contaminated oyster digestive tissues, including 500, 1748, and 1039 up-regulated and 1723, 1242, and 981 down-regulated genes, respectively. In particular, FUT2 and B3GNT4, genes encoding the signaling components of glycosphingolipid biosynthesis, were significantly up-regulated in contaminated samples. In addition, we found up-regulation of some immune- and disease-related genes in the MHC I pathway (PA28, HSP 70, HSP90, CANX, BRp57, and CALR) and MHC II pathway (GILT, CTSBLS, RFX, and NFY), although NoVs did not cause diseases in the oysters. We detected two types of HBGA-like molecules with positive-to-negative ratios similar to type A and H1 HBGA-like molecules in digestive tissues that were significantly higher in norovirus-contaminated than in non-contaminated oysters. Thus, our transcriptome data analysis indicated that a human pathogen (GII.4 Norovirus) was likely concentrated in the digestive tissues of oysters via HBGA-like molecules that were synthesized by the glycosphingolipid biosynthesis pathway. The identified differentially expressed genes also provide potential candidates for functional analysis to identify genes involved in the accumulation of noroviruses in oysters.

Purpose: In a previous study, we identified the Asn450Tyr mutant myocilin gene (Myoc-N450Y) in the pedigree of families with juvenile open angle glaucoma (JOAG), but whether N450Y is a pathogenic mutation remained to be determined. The present study aimed at exploring the role of Myoc-N450Y in primary human trabecular meshwork (HTM) cells.

Methods: Primary HTM cells were infected with lentivirus with wild-type myocilin (Myoc-WT) or Myoc-N450Y. Primary HTM cells overexpressing Myoc-WT or Myoc-N450Y was treated with sodium 4-phenylbutyrate (4-PBA) or not. The secretion and intracellular distribution of Myoc were analyzed with western blotting and immunofluorescence. Expression of endoplasmic reticulum (ER) stress-related proteins was detected with quantitative real-time PCR (qRT-PCR) and western blotting. Cell viability, apoptosis, and expression of the related proteins were examined with Cell Counting Kit-8 (CCK-8), flow cytometry analysis, and western blotting, respectively.

Results: We found that non-secretion of Myoc-N450Y induced ER stress by colocalization with the ER marker calreticulin (CALR), and upregulating the expression of ER stress markers in primary HTM cells. Moreover, overexpression of Myoc-N450Y inhibited the viability and induced apoptosis of primary HTM cells, and inhibition of PI3K/AKT signaling was induced by ER stress. Reduction in ER stress with 4-PBA decreased the level of ER stress markers, promoted secretion, and prevented accumulation of myocilin in the Myoc-N450Y group. Apoptosis was rescued, and inhibition of PI3K/AKT signaling was reversed, after PBA treatment in primary HTM cells with Myoc-N450Y overexpression.

Conclusions: The study results suggest that Myoc-N450Y promotes apoptosis of primary HTM cells via the ER stress-induced apoptosis pathway, in which the PI3K/AKT signaling pathway plays a crucial role.

Objective: To investigate the clinical characteristics, laboratorial and bone marrow pathological features of primary thrombocytopenia (ET) patients with different mutations of CALR, JAK2 and MPL genes.

Methods: The chinical data of 120 cases of ET in Jiangsu provincial people's hospital/ The First Affiliated Hospital of Nanjing Medical University from January 2015 to December 2017 were collected and analyzed, including 76 cases with JAK2 gene mutation, 40 cases with CALR gene mutation, 2 cases with MPL gene mutations, 2 cases without gene mutation.

Results: Among the ET patients, compared with the JAK2 gene mutation, CALR gene mutation showed statistically significant deareament of white blood cells and hemoglobin (P=0.001, P=0.01) and the male platelets in CALR group showed significant increament (P=0.04). Fourthermore, the average number of megakaryocytes and its cluster numbers in each hight power field of vision showed statistically significant decreament in CALR group as compared with JAK2 group (P=0.001, P=0.001), and thrombotic events in CALR group were signicantly lower than those in JAK2 group (7.5% vs 18.4%) (P=0.03).

Conclusion: Mutations of CALR, JAK2 have different clinical characteristics and blood pathological changes of Chinese ET patients, and their clinical significance is worth to explore.

题目: 不同基因突变的中国原发性血小板增多症患者临床特征 及骨髓病理学特点.

目的: 探讨CALR、JAK2、MPL不同基因突变原发性血小板增多症（ET）患者的临床特征、实验室及骨髓病理学特点.

方法: 2015年1月- 2017年12月于我院确诊的ET患者120例，其中JAK2基因突变76例，CALR基因突变40例，MPL基因突变2例，3基因突变阴性患者2例.比较JAK2基因突变ET与CALR基因突变ET临床及实验室特点，采用Nikon cclipsc显微镜进行病理切片观察，每个病理切片取10个不同高倍视野进行巨核计数，巨核细胞集簇数并测量每个高倍视野最大巨核细胞直径.

结果: ET患者中，CALR突变组与JAK2突变组相比较，白细胞数、血红蛋白水平均明显降低（P=0.001，P=0.001），男性血小板数明显升高（P=0.04）；每高倍镜视野中巨核细胞平均值明显降低（P=0.001），平均巨核细胞集簇数明显减少（P=0.001）。CALR突变组血栓事件显著低于JAK2突变组（7.5% vs 18.4%)（P=0.03）.

结论: 中国ET患者中，CALR基因突变者较JAK2基因突变者临床特征和血液病理改变存在差异，临床意义值得探索.

Background: Philadelphia-negative myeloproliferative neoplasms (MPNs) are a group of hematopoietic stem cell disorders that include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). This study examines the driver mutations among patients with MPNs in Kuwait.

Patients and methods: This study was a retrospective review of 942 MPN cases with a driver mutation from July 2007 to June 2019 to examine their demographic, clinical, and laboratory attributes.

Results: The annual incidence of MPNs is 1.6 per 100,000 persons, and ET is the most common subtype. The median age of our cohort was 55 years, and the patients were predominantly male. We found that the most frequent gene mutation of MPNs in our cohort was the JAK2V617F mutation, which was present in 90% of cases, followed by the CALR exon 9, MPLW515L/K, and JAK2 exon 12 mutations. In our cohort, thrombotic events were observed in 18.7% of cases.

Conclusion: Although Philadelphia-negative MPNs are rare hematologic malignancies, thrombosis is a relatively common initial presentation. The JAK2V617F mutation was the driver mutation in the majority of patients with MPN.

Keywords: Essential thrombocythemia; Kuwait; Myelofibrosis; Polycythemia vera; Thrombosis.

Retinal expression of transgenes was examined in four mouse lines. Two constructs were driven by the choline acetyltransferase (ChAT) promoter: green fluorescent protein conjugated to tau protein (tau-GFP) or cytosolic yellow fluorescent protein (YFP) generated through CRE recombinase-induced expression of Rosa26 (ChAT-CRE/Rosa26YFP). Two other constructs targeted inhibitory interneurons: GABAergic horizontal and amacrine cells identified by glutamic acid decarboxylase (GAD65-GFP) or parvalbumin (PV) cells (PV-CRE/Rosa26YFP). Animals were transcardially perfused and retinal sections prepared. Antibodies against PV, calretinin (CALR), calbindin (CALB), and tyrosine hydroxylase (TH) were used to counterstain transgene-expressing cells. In PVxRosa and ChAT-tauGFP constructs, staining appeared in vertically oriented row of processes resembling Müller cells. In the ChATxRosa construct, populations of amacrine cells and neurons in the ganglion cell layer were labeled. Some cones also exhibited GFP fluorescence. CALR, PV and TH were found in none of these cells. Occasionally, we found GFP/CALR and GFP/PV double-stained cells in the ganglion cell layer (GCL). In the GAD65-GFP construct, all layers of the neuroretina were labeled, except photoreceptors. Not all horizontal cells expressed GFP. We did not find GFP/TH double-labeled cells and GFP was rarely present in CALR- and CALB-containing cells. Many PV-positive neurons were also labeled for GFP, including small diameter amacrines. In the GCL, single labeling for GFP and PV was ascertained, as well as several CALR/PV double-stained neurons. In the GCL, cells triple labeled with GFP/CALR/CALB were sparse. In conclusion, only one of the four transgenic constructs exhibited an expression pattern consistent with endogenous retinal protein expression, while the others strongly suggested ectopic gene expression.

The baculovirus expression vector system is widely used for the production of recombinant proteins. However, the yield of membrane-bound or secreted proteins is relatively low when compared with intracellular or nuclear proteins. In a previous study, we had demonstrated that the co-expression of the human chaperones calreticulin (CALR) or β-synuclein (β-syn) increased the production of a secreted protein considerably. A similar effect was also seen when co-expressing insect translation initiation factor eIF4E. In this study, different combinations of the three genes were tested (CALR alone, β-syn + CALR, or β-syn + CALR + eIF4E) to further improve secretory protein production by assessing the expression level of a recombinant secreted alkaline phosphatase (SEFP). An additional 1.8-fold increment of SEFP production was obtained when cells co-expressed all the three "helper" genes, compared to cells, in which only CALR was co-produced with SEFP. Moreover, the duration of the SEFP production lasted much longer in cells that co-expressed these three "helper" genes, up to 10 dpi was observed. Utilization of this "triple-supporters" containing vector offers significant advantages when producing secreted proteins and is likely to have benefits for the production of viral vaccines and other pharmaceutical products.

The baculovirus expression vector system (BEVS) is widely used as a tool for expressing of recombinant proteins in insect cells or larvae. However, the expression level of secretion pathway proteins is often lower than that of cytosolic and nucleus proteins. Thus, we attempted to improve production of secreted proteins by using a secretory alkaline phosphatase-EGFP fusion protein (SEFP)-based bi-cistronic baculovirus vector to identify chaperones that have potential on boosting secreted protein production. As co-expressed SEFP with a chaperone, calreticulin (CALR), it was found that the secreted SEFP enzyme activity can be boosted up to twofold. This result demonstrated the SEFP-based bi-cistronic approach can be used to identify the genes that can enhance secretion protein production in BEVS. Thus, the chaperone activity of α-synuclein (α-syn) and β-synuclein (β-syn) was evaluated in cells co-expressed with SEFP and compared that with CALR by analyzing localization, alkaline phosphatase enzyme activity, and mRNA expression levels of SEFP. Our results showed that SEFP enzyme activity from cells co-expressed with both synuclein proteins can be enhanced up to 2.3-fold and this increment was better than that caused by CALR. Moreover, this enhancement might arise from the transcription enhancement or higher RNA stability. By this novel approach, we provided evidences that α- and β-syn can enhance secretion proteins production in BEVS.

Coronavirus disease (COVID-19) is an infectious disease discovered in 2019 and currently in outbreak across the world. Lung injury with severe respiratory failure is the leading cause of death in COVID-19, brought by severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2). However, there still lacks efficient treatment for COVID-19 induced lung injury and acute respiratory failure. Inhibition of Angiotensin-converting enzyme 2 (ACE2) caused by spike protein of SARS-CoV-2 is the most plausible mechanism of lung injury in COVID-19. We propose two candidate drugs, COL-3 (a chemically modified tetracycline) and CGP-60474 (a cyclin-dependent kinase inhibitor), for treating lung injuries in COVID-19, based on their abilities to reverse the gene expression patterns in HCC515 cells treated with ACE2 inhibitor and in human COVID-19 patient lung tissues. Further bioinformatics analysis shows that twelve significantly enriched pathways (P-value <0.05) overlap between HCC515 cells treated with ACE2 inhibitor and human COVID-19 patient lung tissues, including signaling pathways known to be associated with lung injury such as TNF signaling, MAPK signaling and Chemokine signaling pathways. All these twelve pathways are targeted in COL-3 treated HCC515 cells, in which genes such as RHOA, RAC2, FAS, CDC42 have reduced expression. CGP-60474 shares eleven of twelve pathways with COL-3 with common target genes such as RHOA. It also uniquely targets genes related to lung injury, such as CALR and MMP14. In summary, this study shows that ACE2 inhibition is likely part of the mechanisms leading to lung injury in COVID-19, and that compounds such as COL-3 and CGP-60474 have the potential as repurposed drugs for its treatment.